Food allergen analysis: Considerations for establishing a reference measurement system to implement EU legislation.

Inconsistent quantification results obtained from various analytical methods for food allergen testing hamper an accurate quantitative risk assessment and its regulatory implementation. In order to overcome such problems, a concept aiming at ensuring the comparability of quantitative food allergen measurement results is presented here. It is based on an approach called reference measurement system for food allergens, which uses a commonly agreed reference, namely the 'mass fraction of total protein of the allergenic ingredient in food'. The necessary system components are outlined, consisting of a primary reference measurement method, a certified reference material and a reference laboratory. This metrology-based concept can be applied to quantify various food allergens determined with different analytical procedures. The example of 'milk in cookies' is used to demonstrate the approach.

Assignment of a Reference Value of Total Cow's Milk Protein Content in Baked Cookies Used in an Interlaboratory Comparison.

Interlaboratory comparisons (ILC) in the food allergens field mainly rely on the use of consensus values per applied methodology or even per type of an ELISA test kit. Results suggest good reproducibility; however, possible biases may not be recognized since metrological traceability to an independent reference is lacking. The work presented here utilizes isotope dilution mass spectrometry (IDMS) to assign a reference value of the total cow's milk protein (TCMP) content in a baked cookie and its associated uncertainty. TCMP consists of several individual proteins, of which five (representing 92%) served us as markers for TCMP. Per marker, one to four proteotypic peptides were selected for the quantification. These were synthesized, and the mass fractions of respective reference solutions were determined with peptide-impurity-corrected amino acid analysis to establish traceability to SI units. Stable isotope labelled ("heavy") analogues of the proteotypic peptides were also synthesized and blended with extracts of the test material or the reference solutions for IDMS. Through careful measurement design minimizing biases, well-defined model equations were developed, allowing appropriate estimation of the associated uncertainty. The determined reference value of 11.8 ± 1.1 mg TCMP/kg cookie was used for scoring of a novel ILC.

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept.

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely 'mass of total allergen protein per mass of food'. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors. Graphical abstract.

Determination of tropane alkaloids in cereals, tea and herbal infusions: Exploiting proficiency testing data as a basis to derive interlaboratory performance characteristics of an improved LC-MS/MS method.

Monitoring of tropane alkaloids is regulated in the European Union in cereal-based foods for infants and young children, tea and herbal infusions. The European Commission's Joint Research Centre (JRC) developed an improved LC-MS/MS analytical method using a pentafluorophenyl column, validated it and conducted two proficiency tests targeting these food categories. A subset of the data gathered from laboratories that used the JRC method was additionally exploited to derive interlaboratory performance characteristics. The method showed fit-for-purpose figures of merit. The LOQs for atropine and scopolamine were around 0.4 and 1.2 µg/kg in cereal products, and in tea and herbal infusions, respectively. Uncertainties varied from 15 to 25%. The reproducibility varied from 11 to 38% for scopolamine and from 17 to 44% for atropine at levels ranging from 0.18 to 18.8 and 1.2-54.0 µg/kg, respectively. Recoveries ranged from 71 to 96%. These performance parameters render the method a good candidate for standardisation.

Metabolomics for organic food authentication: Results from a long-term field study in carrots.

Increasing demand for organic products and their premium prices make them an attractive target for fraudulent malpractices. In this study, a large-scale comparative metabolomics approach was applied to investigate the effect of the agronomic production system on the metabolite composition of carrots and to build statistical models for prediction purposes. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) was applied successfully to predict the origin of the agricultural system of the harvested carrots on the basis of features determined by liquid chromatography-mass spectrometry. When the training set used to build the OPLS-DA models contained samples representative of each harvest year, the models were able to classify unknown samples correctly (100% correct classification). If a harvest year was left out of the training sets and used for predictions, the correct classification rates achieved ranged from 76% to 100%. The results therefore highlight the potential of metabolomic fingerprinting for organic food authentication purposes.

Determination of Ochratoxin A in Black and White Pepper, Nutmeg, Spice Mix, Cocoa, and Drinking Chocolate by High-Performance Liquid Chromatography Coupled with Fluorescence Detection: Collaborative Study.

A method validation study for the determination of ochratoxin A in black and white pepper (Piper spp.), nutmeg (Myristica fragrans), spice mix (blend of ginger, turmeric, pepper, nutmeg, and chili), cocoa powder, and drinking chocolate was conducted according to the International Harmonized Protocol of the International Union of Pure and Applied Chemistry. The method is based on the extraction of samples with aqueous methanol, followed by a cleanup of the extract with an immunoaffinity column. The determination is carried out by reversed-phase LC coupled with a fluorescence detector. The study involved 25 participants representing a cross-section of research, private, and official control laboratories from 12 European Union (EU) Member States, together with Turkey and Macedonia. Mean recoveries ranged from 71 to 85% for spices and from 85 to 88% for cocoa and drinking chocolate. The RSDr values ranged from 5.6 to 16.7% for spices and from 4.5 to 18.7% for cocoa and drinking chocolate. The RSDR values ranged from 9.5 to 22.6% for spices and from 13.7 to 30.7% for cocoa and drinking chocolate. The resulting Horwitz ratios ranged from 0.4 to 1 for spices and from 0.6 to 1.4 for cocoa and drinking chocolate according to the Horwitz function modified by Thompson. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, and it conforms to requirements set by current EU legislation.

Intersex related gene expression profiles in clams Scrobicularia plana: Molecular markers and environmental application.

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Intersex in Scrobicularia plana: transcriptomic analysis reveals novel genes involved in endocrine disruption.

Intersex, the appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide, yet the underpinning molecular etiology remains uncharacterized. The presence of intersex has been shown to be a widespread phenomenon in bivalve, S. plana, populations from the southwest coast of the U.K., as well as inducible in an experimental exposure regime using endocrine disrupting compounds (EDCs). Herein, we use the suppressive subtractive hybridization approach to isolate differentially expressed transcripts in S. plana males exhibiting intersex. Transcripts involved in cell signaling, cell cycle control, energy production/metabolism, microtubule assembly, and sperm physiology are all highlighted as differentially expressed in intersex male clams. These provide both an insight into the molecular mechanisms of action involved in the development of intersex, as well as facilitating potential molecular-level "early warning" biomarkers of the condition.

Metabolomic analysis of sex specific metabolites in gonads of the mussel, Mytilus edulis.

Marine mussels have been used as sentinel organisms to monitor exposure to a variety of chemical contaminants, including endocrine disrupting chemicals, in the aquatic environment. Although they are an important species for use in ecotoxicology investigations, information on their reproductive physiology and biochemistry is fragmentary. Mass spectrometry-based profiling techniques are increasingly being used to study how the metabolome of an organism changes as a result of tissue differentiation, disease or in response to environmental stressors. In this study, ultraperformance liquid chromatography-time-of-flight-mass spectrometry (UPLC-TOFMS) was used to investigate sex specific differences in the mussel metabolome in order to further investigate the reproductive physiology of this species. Using this method, a comparison of female and male mantle tissues containing mature gonad, revealed significant differences in glycerophosphatidylcholine (PC) and lysophosphatidylcholine (LPC) metabolites. A number of other unidentified metabolites, including those putatively identified as conjugated sterols, were also differentially expressed between male and female mantle/gonadal tissue.

Two CYP3A-like genes in the marine mussel Mytilus edulis: mRNA expression modulation following short-term exposure to endocrine disruptors.

Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation.

Identification of reproduction-specific genes associated with maturation and estrogen exposure in a marine bivalve Mytilus edulis.

BACKGROUND: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. METHODOLOGY/PRINCIPAL FINDINGS: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. CONCLUSIONS: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.

Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure.

PURPOSE: The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. METHODS: To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. RESULTS: Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. CONCLUSIONS: We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis.

Mytilus edulis were exposed to 17beta-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.

Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis).

Estrogenic contaminants in the aquatic environment are associated with feminisation of male fish, however their effects on some invertebrate species, such as bivalve molluscs, have yet to be characterised. Gametogenesis represents a critical step in the reproductive process and is subjected to hormonal control by serotonin (5-HT), prostaglandins (synthesised by cyclooxygenases-COX) and steroids such as 17beta-estradiol (E2). Here, we examine the responses of 5-HT receptor and COX mRNA expression in mussels, Mytilus edulis, exposed to estrogenic compounds during different stages of their reproductive cycle. In mature mussels, 5-HT receptor mRNA expression decreased following E2 exposure. The opposite trend was observed in mussels at early gametogenesis stages. COX mRNA expression levels at both stages were generally decreased by E2 exposure. Mussels at early gametogenesis stages were also exposed to ethynylestradiol (EE2) and estradiol benzoate (EB) and a significant increase in 5-HT receptor mRNA expression was observed with both xeno-estrogens. COX expression levels were increased with EB exposure but no significant effects were found with EE2 exposure. These results show that the natural estrogen, E2, as well as the synthetic estrogen, EE2, induce alterations, dependent on reproductive stage, in the mRNA expression levels of 5-HT receptor and/or COX in the marine bivalve M. edulis.